Human cells utilize large protein complexes to regulate basic processes that are essential for normal function. A shared feature of these complexes is their dynamic nature: they must undergo structural transitions to mediate their biological functions. Perhaps the most striking example of this is the human Mediator complex, whose structural state is altered upon binding distinct DNA-binding transcription factors, including the p53 tumor suppressor. Remarkably, the structural shifts occur throughout the 1.2 MDa, 26-subunit complex, suggesting a massive re-organization of subunit-subunit architecture. More important, the p53-induced structural shift alters the function of the Mediator complex: p53-bound Mediator activates transcription, whereas activator-free Mediator (which adopts a distinct structural state) does not. The correlation between the p53-induced structural shift and activation of p53 target gene expression suggests the possibility of controlling p53 activity (and its oncogenic potential in p53 gain-of-function cancer cells) by targeting key protein interfaces that are required to propagate the structural shift. Currently, it is not possible to study the dynamic architecture of large complexes such as Mediator due to a lack of appropriate technology. To address this critical unmet need, we developed an integrated chemical control crosslinking-mass spectrometric (CXMS) and computational approach that enables mapping the architecture of large protein complexes. Our strategy centers around an innovative, bi-functional, MS-labile crosslinking reagent called BDRG, coupled with high mass accuracy MS analysis. The objective of this proposal is to assess the feasibility of using BDRG CXMS to map the dynamic architecture of the human Mediator complex. In the Aim we will apply the BDRG CXMS approach to map the subunit architecture of the human Mediator complex in two distinct structural and functional states. Specifically, we will examine activator-free (inactive) Mediator complexes and Mediator complexes bound to the activation domain of the p53 (active). The research is significant because, if successful, the crosslinking data will reveal key control points that represent Mediator subunit-subunit interfaces required for p53-directed structural shifts. Such information could not be readily attained using existing biochemical or biophysical approaches, and would provide new strategies and targets for controlling p53 function in cancer cells (future studies). Furthermore, the results would for the first time elucidate the subunit organization of human Mediator, a genome-wide regulator of transcription. Finally, successful application of the BDRG CXMS technology to the human Mediator complex would firmly establish this approach as an effective means to interrogate structure- function relationships within large, dynamic assemblies. This would open the door for more general application of the technology to other large, cancer-relevant molecular machines, such as DNA-repair complexes or the spliceosome.